Clearing agent and mounting medium for microscopy

ABSTRACT

A clearing agent and mounting solution for microscopy is disclosed comprising (a) trichloroethanol, (b) optionally, trichloroacetic acid, (c) optionally, glycerol and (d) optionally, water, where the refractive index of the solution is greater than or equal to about 1.3810. The solution can further comprise a C1-C6 alcohol, other acids, and/or a stain. The solution can also comprise derivatives and/or analogs of 2,2,2-trichloroethanol and/or trichloroacetic acid. Also disclosed is a method of preparing specimens for microscopy comprising (a) applying a specimen to a microscope slide or a cuvette, (b) applying a quantity of the above solution sufficient to mount the specimen, and (c) optionally applying a cover slip. The solution can be used effectively with stains or dyes, and with fresh, partially dry or dried materials, and for temporary or semi-permanent to permanent mounting. The solution can be used with specimens or tissues/cells/parts originating from animals, poultry, livestock, humans, higher plants, yeasts, molds, microorganisms, insects, mites, or reptiles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under U.S.C. 35 §119(e)of U.S. Provisional Application No. 61/622,210, filed on Apr. 10, 2012,the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention is related to the preparation of specimens for microscopy,and particularly to the development of replacements for chloral hydrateas a clearing agent and mounting medium for microscopy.

BACKGROUND OF THE INVENTION

There are different methods for identification of materials includingmacroscopic, chemical and microscopic identification among others.Microscopic identification is a technique that uses a microscope toidentify characteristic features of living organisms, parts of anorganism, cells or sub-cellular organs, as well as minerals or othernon-living materials. The importance of microscopy resides in theability to clearly identify differences between organisms or their partsby focusing on specific characteristics or diagnostic structures.Microscopy relies on dependable, readily available reagents as clearingagent and mounting solutions, optionally used in conjunction with stainsin order to visualize the materials under the slide.

The general microscopy procedure for specimens derived from livingorganisms comprises mounting a small sample of the tissue to be analyzedin a solvent solution and observing it under the microscope. In manycases the cell contents obscure the tissues, making it difficult toidentify characteristic features. Differences in refractive index withinthe specimen prohibit visualization of deeper visual planes, and occludedetail from observation. In these cases a clearing solution is appliedin order to improve the transparency of the specimen, allowing one tovisualize multiple vertical layers of the specimen without carefulsectioning or remounting. This increased transparency and improvedclarity allows the microscope user to visualize across a full range ofvertical planes in the sample, allowing the user to select interestingfocal planes by adjusting the focus.

A valuable and almost universally used clearing agent for microscopy isacidified chloral hydrate glycerol solution (chloral hydrate solutionacidified with hydrochloric acid), also known as Hertwig's solution.Acidified chloral hydrate solution is used in botanical microscopy,mycology, entomology, histology, mineralogy, food science, qualitycontrol, forensics, nematology, archeology, paleontology, virology,immunology, microscopy including but not limited to differentialinterference contrast microscopy, electron microscopy, fluorescencemicroscopy, confocal microscopy, and other related applications ofmicroscopy and optics. Chloral hydrate when applied to botanical samplesdissolves cellular contents and intercellular substances thus allowingcell walls and shapes of the cells to be easily observed. Chloralhydrate solution has a high refractive index, which improves itstransparency over media with lower refractive indices (such as water).

Many Pharmacopeias contain published protocols for microscopicauthentication analyses of herbal preparations using acidified chloralhydrate as the clearing agent. Consequently, chloral hydrate has becomethe industry standard and an important reagent required on a daily basisfor many laboratories focused on quality assessment of herbal products.For example, in botanical samples, chloral hydrate is used to assist inthe identification of cells with suberized cell walls, fibers (anelongated cell with thick cell walls at maturity that gives strength andsupport to the plant tissue), vessel elements (a tube-like series ofcells with wide lumen and perforated walls), trichomes (hair-likestructures that project from the epidermal surface of the leaves,flowers and stems), stoma (pores in the epidermis of the leaf throughwhich the plant exchanges gases) and pollen.

Chloral hydrate, the key component in acidified chloral hydratesolution, is considered under US law to be a narcotic hypnotic, and assuch is a DEA (Drug Enforcement Administration) scheduled substance,requiring DEA approval and compliance in order to purchase and/orpossess it. This has precluded scientists from being able to purchasethis reagent; and in particular has precluded academic institutions(elementary schools, middle schools, high schools, colleges anduniversities) from purchasing this almost universal reagent that isrequired for preparing slides across all disciplines—from plant science,to biological sciences, to medical sciences and more. Furthermore,maintaining DEA compliance is a costly, tedious, and time-consumingprocess.

Thus, a major disadvantage of using acidic chloral hydrate-glycerolsolution is that chloral hydrate is a controlled substance and requiresa special permit in order to purchase, possess, or use in the US.Therefore cost-effective, readily available and unregulated replacementsfor acidified chloral hydrate solution are needed as clearing andmounting agents for microscopy.

BRIEF SUMMARY OF THE INVENTION

It has now been discovered that aqueous solutions of trichloroethanol,or its derivatives or analogs, with or without other additives, caneffectively replace acidified chloral hydrate solution as a clearing andmounting medium for microscopy.

One embodiment of the present invention is directed to novel chemicalsolutions for clearing and mounting tissues for microscopicidentification which overcomes the above-identified issues withacidified chloral hydrate solution, without sacrificing the quality andresolution of the images observed. Another embodiment of the inventionis directed to methods of preparing specimens for microscopy using theclearing agent and mounting solution.

One embodiment of the invention is directed to a clearing agent andmounting solution including a permanent mount for microscopy comprising:

(a) about 10% to about 97% (v/v) of trichloroethanol;

(b) 0% to about 50% (v/v) of trichloroacetic acid;

(c) 0% to about 50% (v/v) of glycerol; and

(d) optionally, water;

-   wherein the refractive index of said solution is greater than or    equal to about 1.3810.-   Preferably the refractive index is about 1.3810 to about 1.4880.-   Preferably trichloroethanol is present in about 34.5% to about 97%;    more preferably in about 55% (v/v). Preferably trichloroacetic acid    and glycerol are present in 0% to about 5%, and 0% to about 12.5%,    respectively.

In one particular embodiment, the clearing agent and mounting solutioncontains trichloroethanol in about 56.25% (v/v), trichloroacetic acid inabout 1.25% (v/v), glycerol in about 12.5% (v/v), and water in about 30%(v/v).

The clearing agent and mounting solutions can further comprise one ormore of the following:

-   1. a C1-C6 alcohol in about 5-85% (v/v), for example methanol and/or    ethanol in about 25-30%;-   2. an inorganic acid selected from the group consisting of    hydrochloric acid, sulfuric acid, phosphoric acid, and mixtures of    two or more thereof;-   3. a stain or dye selected from the group consisting of carmine,    carmine, crystal violet, gram stain, aniline blue, phoroglucinol,    lactophenol, sudan IV, iodine/potassium iodide stain, eosin and    fuchsin;-   4. a dissolved plastic polymer or resin;-   5. monomeric units, polymerization of which stabilizes a mounting    medium and/or specimen;-   6. one or more C1-C12 organic acids, which can be selected from the    group consisting of acetic acid, formic acid, lactic acid and citric    acid;-   7. a chemical preservative, which preserves the integrity of the    specimen or extends its working and viewable lifetime;-   8. a salt solution or buffer, which can be selected from the group    consisting of phosphate, citrate, acetate, tris or other    water-soluble buffers;-   9. an alkaline hydroxide base, which can be selected from the group    consisting of sodium hydroxide, calcium hydroxide, potassium    hydroxide, magnesium hydroxide and lithium hydroxide;-   10. a compound selected from the group consisting of ethylene    glycol, polyethylene glycols and derivatives thereof;-   11. polyvinylpyrrolidone dissolved in an organic solvent selected    from the group consisting of ethanol, methanol, isopropanol,    acetone, and mixtures of two or more thereof;-   12. compounds selected from the group consisting of derivatives and    analogs of 2,2,2-trichloroethanol, and derivatives and analogs of    2,2,2-trichloroacetic acid;-   13. acidic and/or basic additives to alter pH and/or the salt    concentration of the solution;-   14. compounds to influence the digestive capacity of the reagent;-   15. a solidification aid selected from the group consisting of    polyethylene glycol, polyamide resin, polyvinylpyrrolidone,    polyvinylalcohol and mixtures of two or more thereof;-   16. an aromatic solvent selected from the group consisting of    xylene, toluene, other benzene derivatives and mixtures of two or    more thereof;-   17. dimethylsulfoxide;-   18. an oil selected from the group consisting of cedar oil, pine    oil, peanut oil, other plant-derived oils and mixtures of two or    more thereof;-   19. a dried exudate obtained from the stems and branches of trees    and/or plants containing saccharide-based gums, such as Arabic gum;-   20. a carbohydrate selected from the group consisting of mono-, di-    and poly-saccharide forms of C4-C6 carbohydrates and mixtures of two    or more thereof;-   21. an aqueous solution of an amino acid or mixtures of two or more    amino acids.

Another embodiment of the invention is directed to a clearing agent andmounting solution for microscopy comprising:

-   (a) about 10% to about 97% (v/v) of a trichloroethanol derivative or    analog;-   (b) 0% to about 50% (v/v) of a trichloroacetic acid derivative or    analog;-   (c) 0% to about 50% (v/v) of glycerol;-   (d) optionally, water; and-   (e) optionally, 5-85% (v/v) of a C1-C6 alcohol;    wherein the refractive index of said solution is greater than or    equal to about 1.3810; wherein said trichloroethanol derivative or    analog is selected from the group consisting of mono- and    poly-halogenated branched and unbranched alcohols, diols, glycol    aldehydes, aldehyde-hydrates, hemi-acetals, acetals, ketals,    aminals, hemi-aminals of at least 2 carbon units, and polymeric    embodiments thereof; wherein the branches comprise mono- or    poly-halogenated aliphatic or aromatic groups containing hydroxyl,    amino, ether, carboxyl, carboxyamido, carbonate, carbamyl,    carbonyl-chloride, polyethylene-glycol, or aminoethanol groups.

Another embodiment of the invention is directed to a clearing agent andmounting solution for microscopy comprising:

(a) about 10% to about 30% (v/v) of trichloroacetic acid;

(b) about 10% to about 30% (v/v) of sodium hydroxide; and

(c) optionally, water,

wherein the refractive index of said solution is greater than or equalto about 1.3810.

A further aspect of the invention is directed to a method of preparingspecimens for microscopy comprising:

-   -   (a) applying a specimen to be examined to a microscope slide or        a cuvette;    -   (b) applying a sufficient quantity of an inventive clearing        agent and mounting solution to mount said specimen; and    -   (c) optionally, applying a cover slip.        1-100 drops of the clearing agent and mounting solution can be        applied.

In another embodiment, the method of preparing a specimen for microscopycomprises:

-   -   (a) soaking a specimen in sufficient quantity of an inventive        clearing solution for at least 1 minute to about 12 months to        provide a cleared specimen,    -   (b) applying the cleared specimen to a microscope slide,        cuvette, or well for observation, and    -   (c) optionally, applying a cover slip.

Alternatively, after soaking the specimen, the cleared specimen can beapplied to a resin which will solidify, and then be cast into a solidfor examination or indefinite storage.

Alternatively, after soaking the specimen, a solution of 5-20%polyvinylpyrrolidone in methanol can be applied to the cleared specimen,which is optionally, dried at 50-90° C. until hardened.

Alternatively, after soaking the specimen, a stain or dye can be appliedto the cleared specimen to provide a cleared stained specimen wherebythe features of the specimen are selectively highlighted; the clearedstained specimen can be applied to a microscope slide, cuvette, or wellfor observation, and optionally covered with a cover slip. The stain ordye can be a fluorescent stain or dye, so that the cleared stainedspecimen can be visualized using a fluorescent and/or epifluorescentand/or confocal microscope

Another aspect of the invention is directed to a method of preparing aspecimen for spectrophotometric analysis comprising:

-   -   (a) applying a specimen to a cuvette; and    -   (b) applying a sufficient quantity of an inventive clearing        agent and mounting solution to mount said specimen.

Another aspect of the invention is directed to use of an inventiveclearing agent and mounting solution to remove pigment, dye, stain, orcolor from a specimen.

A further aspect of the invention is directed to use of an inventiveclearing agent and mounting solution to remove excess stain or dye andto increase the contrast of particular structures and/or organismswithin a specimen.

Another aspect of the invention is directed to use of an inventiveclearing agent and mounting solution to increase the transparency of aspecimen and to allow multiple vertical planes to be visualized withoutthe need to section, remount, or further modify the specimen.

Yet another aspect of the invention is directed to use of an inventiveclearing agent and mounting solution to simultaneously dehydrate,depigment, and clear specimens for microscopic and/or visual analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a microscopic view of characteristic annular vesselelements and fibers of ginger, Zingiber officinale Roscoe(Zingiberaceae); comparison of acidified chloral hydrate solution versusExample 1 solution.

FIG. 2 shows a microscopic view of abundant starch grains of ginger,Zingiber officinale Roscoe (Zingiberaceae); comparison of acidifiedchloral hydrate solution versus Example 1 solution.

FIG. 3 shows a microscopic view of ginger epidermis and parenchymacells, Zingiber officinale Roscoe (Zingiberaceae); comparison ofacidified chloral hydrate solution versus Example 1 solution.

FIG. 4 shows a microscopic view of fragments of epidermis over leafveins, Mate (leaves), Ilex paraguariensis (Aquifoliacea); comparison ofacidified chloral hydrate solution versus Example 1 solution.

FIG. 5 shows a microscopic view of the upper epidermis underlyingpalisade cells, Mate (leaves), Ilex paraguariensis (Aquifoliacea);comparison of acidified chloral hydrate solution versus Example 1solution.

FIG. 6 shows a microscopic view of fragments or groups of pericycle offibers, Mate (leaves), Ilex paraguariensis (Aquifoliacea); comparison ofacidified chloral hydrate solution versus Example 1 solution.

FIG. 7 shows a microscopic view of the lower epidermis showingcharacteristic anomocytic stomata, Mate (leaves), Ilex paraguariensis(Aquifoliacea); comparison of acidified chloral hydrate solution versusExample 1 solution.

FIG. 8 shows a microscopic view of rounded or elliptical pollen grainswith three germinal pores, exine (outermost cell wall of pollen grain)dentate spinose, Safflower (flower), Carthamus tinctorius L.(Asteraceae); comparison of acidified chloral hydrate solution versusExample 1 solution.

FIG. 9 shows a microscopic view of laticiferous ducts (tubular cellscontaining latex fluid) with a reddish-brown secretion next to vesselselements, Safflower (flower), Carthamus tinctorius L. (Asteraceae);comparison of acidified chloral hydrate solution versus Example 1solution.

FIG. 10 discloses the chemical structures of analogs of2,2,2-trichloroethanol useful for preparation of clearing agent andmounting solutions of the invention.

FIG. 11 displays fresh whole mounted specimens cleared with Example 1clearing agent and mounting solution. A-B: Basil leaf; C-F: Oreganoleaf; G-H: Arabidopsis thaliana root.

FIG. 12 shows a red alga Polysiphonia sp gametophyte showing a secondarybranch forming off the main axis, cleared with Example 1 clearing agentand mounting solution.

FIG. 13 shows a roundworm free living nematode Panagrellus redivivus(Animalia) anterior end showing internal structures, cleared withExample 1 clearing agent and mounting solution.

FIG. 14 shows a small aquatic crustacean Daphnia sp. (Animalia) anteriorend showing internal structures, cleared with Example 1 clearing agentand mounting solution.

FIG. 15 shows a characteristic Drosophila melanogaster (Animalia)compound eye, cleared with Example 1 clearing agent and mountingsolution.

FIG. 16 shows a dorsal view of the head of Drosophila melanogaster(Animalia) showing compound eye, antenna and mouth parts, cleared withExample 1 clearing agent and mounting solution.

FIG. 17 shows the fungus Penicillium sp. conidiophores with a chain ofconidia (asexual spores) at the end, cleared with Example 1 clearingagent and mounting solution.

FIG. 18 shows basil downy mildew (Peronospora belbahrii), stained withiodine solution and sulfuric acid, cleared with Example 1 clearing agentand mounting solution.

FIG. 19 shows a roundworm Panagrellus redivivus (Animalia.) stained withfuchsine, cleared with Example 1 clearing agent and mounting solution.

FIG. 20 shows characteristic starch grains of Ginger (Zingiberofficinale) stained with iodine solution, cleared with Example 1clearing agent and mounting solution.

FIG. 21 shows characteristic lignified fibers of Prunus africana stainedwith phloroglucinol/HCl solution, cleared with Example 1 clearing agentand mounting solution.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

With this invention, it has now been discovered that aqueous solutionsof trichloroethanol, or its derivatives or analogs, with or withoutother additives, can effectively replace acidified chloral hydratesolution as a clearing and mounting medium for microscopy for bothliving and nonliving organisms and nonliving materials. One embodimentof the present invention is a substitute for acidified chloral hydrateglycerol solution, wherein the solution components, for exampletrichloroethanol, are inexpensive, easy to acquire, do not require a DEAlicense to possess and use, and greatly reduce the risk involved inoperations which formerly depended on the use of chloral hydrate. Theinventive solutions possess a high refractive index (greater than orequal to about 1.3810; preferably about 1.3810 to about 1.4880; mostpreferably about 1.4315 to about 1.4880), preferably higher thanacidified chloral hydrate solution (1.4280), which results in clearer,and equivalent or higher quality viewing under a microscope. Highrefractive indices are required for clear viewing of objectives inmicroscopy, as materials with a high refractive index are moretransparent. As a reference, the refractive index of borosilicate(Pyrex) glass is 1.470. Thus, several of the embodiments of the clearingagent and mounting solution of the invention have refractive indiceshigher than that of glass. It has been discovered that trichloroethanol,or its derivatives or analogs, admirably meet the above-identifiedcriteria as replacements for chloral hydrate.

Use of the clearing agent and mounting solution of the invention helpsto macerate and digest clusters of cellular material, and helps toclarify and increase transparency of those tissues, minerals, elementsof interest in microscope slides. This solution is an effectiveimmersion medium, and useful in all types of fixative preparations andas an effective dehydration agent. The clearing compound and/or itsderivatives can also be used as a semi-permanent or permanent mount,allowing one to visualize specimens days or even months later. Thisclearing compound and/or its derivatives can also be used with anystains, allowing one to further visualize specimens and componentswithin specimens.

Description of the Clearing Agent and Mounting Solutions andApplications

This invention encompasses the identification of chemical compounds thathave not heretofore been used in microscopy applications. In oneembodiment, the clearing agent and mounting solution of the inventioncomprises any concentration of 2,2,2-trichloroethanol, or derivatives oranalogs thereof, and/or 2,2,2-trichloroacetic acid, or derivatives oranalogs thereof in water and/or glycerol and/or alcohol solution, mixedfor the purpose of clearing and/or mounting media for microscope/opticaluse.

For the purposes of the present invention, the term “derivative” means achemical compound which still retains the parent structure as asubstructure, and can be chemically derived from the parent (e.g.,trichloroethanol or trichloroacetic acid). For example, with2,2,2-trichloroethanol as the parent, a derivative would retain the2,2,2-trichloroethoxy substructure, such as is found in thecorresponding acetate (1-acetoxy-2,2,2-trichloroethane, alternativelynamed 2,2,2-trichloroethylacetate) or the methyl ether(1-methoxy-2,2,2-trichloroethane) derivatives.

Also for the purposes of the present invention, the term “analog” meansa chemical compound in which the core structure of the parent is changedor further substituted, as is commonly known in the medicinal chemistryarts. For example, the change can include replacement of atoms or groupswith other atoms or groups (isosteres). Examples of analogs of2,2,2-trichloroethanol are displayed in FIG. 10.

In one preferred embodiment, the clearing agent and mounting solution ofthe invention comprises trichloroethanol and/or its analogs orderivatives. In another preferred embodiment, the clearing agent andmounting solution comprises both trichloroethanol and/or its derivativesor analogs, and trichloroacetic acid and/or derivatives or analogs. Oneparticularly preferred embodiment of the invention is directed to aclearing agent and mounting solution for microscopy comprising:

(a) about 10% to about 97% (v/v) of trichloroethanol;

(b) 0% to about 50% (v/v) of trichloroacetic acid;

(c) 0% to about 50% (v/v) of glycerol; and

(d) optionally, water;

wherein the refractive index of said clearing agent and mountingsolution is greater than or equal to about 1.3810, preferably about1.3810 to about 1.4880, most preferably about 1.4315 to about 1.4880.The clearing agent and mounting solution can optionally further containa C1-C6 alcohol, another acid, such as hydrochloric acid, and/or a dyeor stain for visualizing various components of the specimen. Preferablytrichloroethanol is present in about 34.5% to about 97%. Preferablytrichloroacetic acid is present in 0% to about 5%. Preferably glycerolis present in about 0% to about 12.5%. The glycerol can be supplementedor replaced entirely with one or more compounds selected from the groupconsisting of ethylene glycol, polyethylene glycols and derivativesthereof.

Preferably the clearing agent and mounting solution comprises about34.5% to about 97% trichloroethanol, 0% to about 5% trichloroaceticacid, and 0% to about 12.5% glycerol. In one particularly preferredembodiment, the clearing agent and mounting solution containstrichloroethanol in about 56.25% (v/v), trichloroacetic acid in about1.25% (v/v), glycerol in about 12.5% (v/v), methanol in about 25%, andwater in about 5% (v/v).

A further embodiment of the clearing agent and mounting solution of theinvention comprises trichloroacetic acid and/or derivatives withouttrichloroethanol and/or derivatives. One preferred clearing agent andmounting solution consists of an aqueous glycerol solution oftrichloroethanol and trichloroacetic acid (Example 1). Thetrichloroacetic acid can be supplemented or replaced entirely withhydrochloric acid and/or sulfuric acid. One preferred clearing agent andmounting solution consists of an aqueous glycerol solution oftrichloroethanol and hydrochloric acid. The 2,2,2-trichloroethanolanalog, 2-chloroacetaldehyde dimethyl acetal, FIG. 10, also serves as aneffective clearing agent. The clearing agent and mounting solution caninclude derivatives of 2,2,2-trichloroethanol and/or2,2,2-trichloroacetic acid created by adding/adjusting the concentrationof buffer, and/or acidic/basic additives intended to alter pH and/orsalt concentration of the solution; and/or compounds toincrease/decrease the digestive capacity of the reagent; and/or anyadditives intended to preserve specimens/samples; and/or any additivesintended for dying or staining applications.

With regard to the alcohol (other than trichloroethanol or derivatives),the clearing agent and mounting solutions can comprise one or more C1-C6alcohols, for example ethanol, 1-propanol, 2-propanol or t-butanol.

The clearing agent and mounting solutions can also further comprise: adissolved plastic polymer, such as polyvinylpyrrolidone, polypropylene,polyethylene, polyether, polyamide; and/or

monomeric units which are subsequently polymerized in order to stabilizea mounting medium and/or specimen; and/or

one or more C1-C12 organic acids, such as formic acid, acetic acid,lactic acid, ascorbic acid, gallic acid, benzoic acid, toluic acid,p-hydroxybenzoic acid or citric acid; and/or

a chemical preservative; and/or a salt of phosphate, citrate, acetate,tris, or other water-soluble buffers; the buffer can be added as anaqueous solution; and/or alkaline hydroxide base, such as sodiumhydroxide, calcium hydroxide or lithium hydroxide; the base can be addedas an aqueous solution. The inventive clearing agent and mountingsolution and/or its derivatives and/or its analogs can also be used withcommercial and noncommercial stains just as effectively as currentclearing agents.

The present invention also encompasses various derivatives and/oranalogs of 2,2,2-trichloroethanol including mono- or poly-halogenatedbranched or unbranched alcohols, diols, glycols, aldehydes,aldehyde-hydrates, hemi-acetals, acetals, ketals, aminals, andhemi-aminals of at least 2 carbon units, where branches are defined asany mono- or poly-halogenated aliphatic or aromatic side chainscontaining hydroxyl, amino, ether, carboxyl, carboxyamido, carbonate,carbamyl, carbonyl-chloride, polyethyleneglycol, or aminoethanol groups,and any polymeric embodiment of such derivatives.

Derivatives and analogs of 2,2,2-trichloroacetic acid include mono- orpoly-halogenated branched or unbranched carboxylic acids, carbamates,amides, and carbonates of at least 2 carbon units, where branches aredefined as any mono- or poly-halogenated aliphatic or aromatic sidechains containing hydroxyl, amino, ether, carboxyl, carboxyamido,carbonate, carbamyl, carbonyl-chloride, polyethylene-glycol, oraminoethanol groups, and any polymeric embodiment of such derivatives.

Thus, a further embodiment of the invention is directed to a clearingagent and mounting solution for microscopy comprising:

(a) about 10% to about 97% (v/v) of a trichloroethanol derivative oranalog;

(b) 0% to about 50% (v/v) of a trichloroacetic acid derivative oranalog;

(c) 0% to about 50% (v/v) of glycerol; and

(d) optionally, water;

wherein the refractive index of said solution is greater than or equalto about 1.3810; wherein said trichloroethanol derivative or analog isselected from the group consisting of mono- and poly-halogenatedbranched and unbranched alcohols, diols, glycols, aldehydes,aldehyde-hydrates, hemi-acetals, acetals, ketals, aminals, andhemi-aminals of at least 2 carbon units, and any polymeric embodiment ofsuch derivatives; where branches are defined as any mono- orpoly-halogenated aliphatic or aromatic side chains containing hydroxyl,amino, ether, carboxyl, carboxyamido, carbonate, carbamyl,carbonyl-chloride, polyethylene-glycol, or aminoethanol groups.

Another embodiment of the invention is directed to a method of preparinga specimen for microscopy comprising:

-   -   (a) applying a specimen to be examined to a microscope slide,        well, or cuvette;    -   (b) applying a sufficient quantity of the clearing agent and        mounting solution of claim 1 to clear and mount said specimen;        and    -   (c) optionally, applying a cover slip.        1-100 drops, preferably about 2 drops of the clearing agent and        mounting solution are applied to the specimen on the slide in        order to fix/mount the specimen. The specimen can be further        protected with a cover slip.

A related embodiment is directed to a method of preparing a specimen forspectrophotometric analysis comprising:

-   -   (a) applying a specimen to a cuvette or well; and    -   (b) applying a sufficient quantity of the clearing agent and        mounting solution of claim 1 to clear said specimen (1-10×        volume of sample).

As discussed above the present invention is directed to the use ofnon-chloral hydrate clearing and mounting compounds of appropriaterefractive index for novel applications in microscopy. Given the currentshortage and limitation of access to the commercial universally-usedclearing agent (acidified chloral hydrate), the present inventionprovides to consumers and the general public a method that can replacethe currently used clearing agent, and provides a method accessible tothose who are no longer able to purchase the regulated compound chloralhydrate. Thus, the clearing reagents and methods of the presentinvention are of immediate commercial value and of significant impactbecause both scientists and manufacturers have been seeking to find areplacement for chloral hydrate in microscopy and other opticalapplications. The clearing reagents and methods of the present inventionare also of immediate commercial value and of significant impact becausethey can be used with any stains as well as for semi-permanent andpermanent mounting.

Optical Properties

One embodiment of the invention comprises a solution which increases theapparent transparency of an objective in microscopy/optical techniquesby increasing the refractive index of the medium in which said objectiveis suspended/immersed. Refractive index of a material is a dimensionlessquantity which represents the way light propagates through the material.The refractive index is defined as the factor by which the wavelengthand the velocity of the radiation with respect to in a vacuum. Therefractive index of a material is closely related to its dielectricconstant, and therefore to its transparency. The refractive index n of amaterial is given by the following equation:

${n = \sqrt{\frac{\sqrt{\varepsilon_{1}^{2} + \varepsilon_{2}^{2}} + \varepsilon_{1}}{2}}},$where ∈₁ and ∈₂ represent the real and imaginary parts of the dielectricconstant, respectively. Materials which have a high dielectric constantcontain multiple lone pairs of electrons and/or electronegativeelements, which give them a high degree of polarizability, the propertywhich is expressed by the dielectric constant. A high degree ofpolarizability allows for an electromagnetic wave to propagate easilythrough the material, since as the electromagnetic wave propagatesthrough the material, it will induce a localized electromagnetic field.It is useful to use the analogy of waves through liquids, the lessviscous and easier the liquid is to move (by analogy related to higherpolarizability), the easier a wave can propagate through without losingenergy from absorption. In electromagnetic waves, a high polarizabilitycorresponds to this “easier movement” of the wave through the material,which results in less absorption of the wave by the material. Therefore,materials with high dielectric constants will have a low degree ofabsorption, and therefore a high degree of transparency, as theobjective light will make it through the material without absorptionloss. Effectively, the photons of light can escape more unscathed thanthey would in a material with a lower dielectric constant. And sincedielectric constant is related to refractive index as shown above,materials with a high refractive index will also be highly transparent,and therefore of great use in microscopy/optical techniques.

Refractive indices of clearing agent and mounting solutions of theinvention are disclosed in Table 1.

TABLE 1 Refractive indices of various clearing agent and mountingsolutions of the invention. Refractive Clearing Agent and MountingSolution Formulation¹ Index TCE-34.5%; Glyc-12%; HCl (34%)-1.5%;H₂O-28%; MeOH-24% 1.4155 TCE-45%; Glyc-10%; HCl-1.25%; H₂O-23.75%;MeOH-20% 1.4310 TCE-27.4%; Glyc-8.3%; TCAA-0.7%; MeOH-14.2%; LA-49.4%1.4370 TCE-56.25%; Glyc-12.5%; TCAA-1.25%; H₂O-30% 1.4315 TCE-27.4%;Glyc-8.3%; TCAA-0.7%; MeOH-14.2%; DMSO-49.4% 1.4640 TCE-94%; TCAA-5%;NaOH-1% 1.4880 TCE-91%; Glyc-3.3%; TCAA-0.3%; MeOH-5.4% 1.4875TCE-27.4%; Glyc-8.3%; TCAA-0.7%; MeOH-14.2%; Tol-49.4% 1.4770 TCAA-6%;NaOH-4%; H₂O-90% 1.4280 TCE-97%; TCAA-3% 1.4885 TCE-63.0%; Glyc-13.2%;TCAA-1.1%; MeOH-22.7% 1.4565 TCE-27.4%; Glyc-8.3%; TCAA-0.7%; MeOH-63.6%1.3895 TCE-27.4%; Glyc-8.3%; TCAA-0.7%; MeOH-14.2%; CWO-49.4% 1.4790TCE-54.8%; Glyc-16.6%; TCAA-1.4%; MeOH-27.2% 1.4450 ¹TCE =trichloroethanol, TCAA = trichloroacetic acid, Glyc = glycerol, MeOH =methanol, NaOH = sodium hydroxide, LA = lactic acid, DMSO =dimethylsulfoxide, Tol = toluene, CWO = cedar wood oil. All percentagesare v/v.

A number of analog structures have been provided which can be used as asubstitute for trichloroethanol (FIG. 10). 2-chloroacetaldehyde dimethylacetal has been shown to be effective at the same concentrations astrichloroethanol, although the refractive index is only 1.3810, at thelow end of the desired range. One way to achieve the desired refractiveindex is to incorporate a clearing agent having one or more halogens (F,Cl, Br, I) in a carbon skeleton which also contains a water solubilizinggroup capable of hydrogen bonding. For example, the carbon skeletons canbe selected from any mono- or poly-halogenated branched or unbranchedalcohol, diol, glycol, aldehyde, aldehyde-hydrate, hemi-acetal, acetal,ketal, aminal, or hemi-aminal of C1-C20 family, where branches aredefined as any mono- or poly-halogenated aliphatic or aromatic sidechains containing hydroxyl, amino, ether, carboxyl, carboxyamido,carbonate, carbamyl, carbonyl-chloride, polyethylene-glycol, oraminoethanol groups, and any polymeric arrangement of such derivatives.

In comparative qualitative examinations, the clearing agent and mountingsolutions of the invention perform as well as or better than acidifiedchloral hydrate, which is a DEA scheduled substance under US law,requiring DEA approval and compliance in order to purchase, possess oruse the compound. In quantitative examinations, the clearing agent andmounting solution of the invention has matched or outperformed thechloral hydrate-based solutions, and exhibited a refractive indexgreater than chloral hydrate solution, the universal standard. Thisinvention can be used as a clearing agent and mounting solution toidentify the same anatomical characteristics or diagnostic features thatare employed for the identification of different plant, microbial,animal, and earth science materials, without losing clarity, definitionor resolution of the objective structures.

For example the clearing agent and mounting solutions of the inventionare useful for microscopic identification of different plants, plantparts, animals and microbial materials. The clearing agent and mountingsolutions can be used with any living organisms such animals, fungi,protists and bacteria and even with blood and plasma samples, as amounting medium in microscopy and/or other optical techniques withapplications in forensics, and biology and earth sciences. The inventivesolutions can be used to clear specimens, rendering them transparent;these specimens can later be differentially stained and high qualityimages obtained. The inventive clearing agent and mounting solutions andsemi permanent mounting media can also be used with non-livingmaterials, including but not limited to soil particles and geologicalsamples.

The inventive clearing agent and mounting solutions are useful not onlyfor botanical microscopy but also for mycology, entomology, histology,food science, quality control (identification of living organisms formanufacture of pharmaceuticals, excipients, dietary products,adulterations, misidentifications, contaminations), forensics,nematology, virology, immunology, mineralogy, microscopy including butnot limited to differential interference contrast microscopy, electronmicroscopy, and other related applications of microscopy and optics.

For example, one application of the inventive clearing and mountingsolution is in quality assessment of commercial herbal products. It hasalso been determined that the inventive solutions are useful forclearing whole mounted dried, partially dry and fresh materials. Forexample, in basil, the oil glands, epidermis with stomata and underlyingpalisade cells could be observed (FIG. 11 A, B). In oregano, theepidermis over the vein with covering trichomes, capitates and peltateoil glands was distinguished (FIG. 11 C-F). Details of the cellularorganization of the root apical meristem in Arabidopsis thaliana can beobserved after clearing with this invention (FIG. 11 G-H). In addition,a number of other herbs and spices (dry samples and whole tissues) wereanalyzed subsequently using the invention as clearing reagent withcomparable results.

The solutions of the invention penetrate into tissues and render themmore transparent, as does acidified chloral hydrate solution. Aftertreatment with a clearing agent of the invention, samples are cleared,which allows internal as well as surface details to be easilyidentified. This feature is most significant when it is used with wholemount tissues in which different layers of the transparent tissues areobserved without the need for sectioning or remounting. Clear tissuesalso allow for staining techniques to more effectively highlightdiagnostic features in only one single step without requiringdehydration of the tissues or pre-treatment of the tissues (FIG. 11).

The inventive solutions are also effective to clear protists, animalsincluding but not limited to red algae (FIG. 12), round worms(Panagrellus redivivus, FIG. 13), water fleas, Daphnia sp (FIG. 14), andfruit flies (Drosophila melanogaster, FIGS. 15 and 16).

In order to identify different components in the cells, quite frequentlytissues are stained. There are several staining combinations availableto enhance the details between different components of the cells. Stainsare selected to provide the maximum contrast between particularstructures based on their chemical composition. Much of the success ofthe images obtained after staining is due to the clearing agent used asa pretreatment. The solutions of the present invention also have nointerference with the major stains used for particular structures. Asexamples, penicillium sp was first cleared with an inventive clearingagent and then stained with a solution of aniline blue in lactic acid(FIG. 17), downy mildew became dark brown after staining with iodinesolution and sulfuric acid (FIG. 18), the round worm Pangrellusredivivus stained pink/red with fuchsine (FIG. 19), starch grains werestained black with iodine solution in ginger rhizome dry samples (FIG.20) and sclereids fibers were stained red with phloroglucinol/HClsolution (FIG. 21).

EXAMPLES

General procedures

Preparation of Plant Specimens

Plant materials were dried or used fresh. Dried plant materials wereground to a fine powder using a commercial coffee grinder.

Mounting of Specimens for Microscopy

A small quantity of fine powder material was spread on a microscopeslide, and mounted with two drops acidified chloral hydrate solution(control), or mounted with two drops of the clearing agent and mountingsolution of Example 1 and a cover slip was attached. Then the slide washeated on a hot plate (medium temperature) for 30-60 sec until theclearing agent and mounting solution boiled. The microscopic analysiswas conducted using a Nikon eclipse 80i microscope, with the imagingsoftware NIS D 3.00 SP7. Differences or similarities in diagnosticfeatures or characteristics for each sample were recorded. Freshspecimens were submerged in the clearing agent and mounting solutionuntil they were transparent, usually taking around 20-30 minutesdepending the thickness of the material. Larger samples may require upto 2-3 days. Once the material was cleared, it was mounted on amicroscope slide with one or two drops of this invention and a coverslip was added. Fresh specimens were cleared and transparent, allowingthe visualization of deeper layers of tissues without losing clarity.

For staining the materials, first the material was cleared as mentionedabove, cover slip was removed, one or two drops of the stain was addedwaited for few minutes and finally the cover slip was applied.

Example 1 Preparation of a Clearing Agent and Mounting Solution of theInvention

4.5 mL of 2,2,2-trichlorethanol (Sigma-Aldrich, 99%, reagent grade) wasthoroughly mixed with 1.0 mL of glycerol. To this homogenous solutionwas added 2.5 mL of a 4% (m/v) aqueous solution of 2,2,2-trichloroaceticacid, at which time cloudiness was observed. The solution was mixedthoroughly until completely clear, about 5 minutes.

Example 2 Ginger (Rhizome), Zingiber officinale Roscoe (Zingiberaceae);FIGS. 1-3

Powdered ginger samples are characterized by numerous fragments ofisodiametric thin-walled parenchyma cells containing starch granules;fragments of thin-walled fibers with oblique slit-like pits; fragmentsof scalariform, reticulate, and spiral vessels, thin-walled cells withsuberized radial walls; numerous starch granules with various forms suchas simple, flat, oval, oblong with terminal protuberance.

In ground ginger samples, fragments of parenchyma cells, cell withcircular striations (cork cells), fibers accompanied by vessels andabundant starch grains the compact epidermal cells with sharp edges canbe observed using the clearing agent and mounting solution of theinvention. There was no difference in the structures observed betweenthe inventive and standard clearing agent and mounting solutions.

FIG. 1 shows a microscopic view of characteristic annular vesselelements and fibers of ginger: FIG. 1A, ginger characteristic annularvessel element and fibers using acidified chloral hydrate solution; FIG.1B, ginger characteristic annular vessel element with fibers andabundant starch grains attached to the fibers using clearing agent andmounting solution of Ex 1.

FIG. 2 shows a microscopic view of abundant starch grains of ginger.More starch grains can be observed using the clearing agent and mountingsolution of Example 1, versus acidified chloral hydrate solution: FIG.2A, ginger sample with characteristic abundant starch granules, mostlysimple, using acidified chloral hydrate solution; FIG. 2B, ginger samplewith abundant starch grains using clearing agent and mounting solutionof Ex 1.

FIG. 3 shows a microscopic view of ginger epidermis and parenchymacells. There is no difference in the structures observed using theinventive solution, versus acidified chloral hydrate solution: FIG. 3A,group of compact epidermal cells using acidified chloral hydratesolution; FIG. 3B, group of compact epidermal cells using clearing agentand mounting solution of Ex 1.

Example 3 Mate (Leaves), Ilex paraguariensis (Aquifoliacea); FIGS. 4-7

Ground Ilex leaves are characterized by upper epidermis composed bypolygonal cells with unevenly thickened walls. Lower epidermis cells aresmaller than those of the upper epidermis, and thinner cell walls,cuticular striations are well marked. Stomata anomocytic (epidermalcells surrounding the guard cell pair are not morphologically distinctfrom the other epidermal cells). Groups of lignified fibers are visible.

FIG. 4 shows a microscopic view of fragments of epidermis over leafveins. The upper epidermis is composed of polygonal cells with unevenlythickened walls. Stomata are absent in the upper epidermis. There are nodifferences observed using the inventive solution, versus acidifiedchloral hydrate solution: FIG. 4A, fragment of polygonal cells of theupper epidermis over the vein using acidified chloral hydrate solution;FIG. 4B, fragments of polygonal cells of the upper epidermis over thevein using clearing agent and mounting solution of Ex 1.

FIG. 5 shows a microscopic view of the upper epidermis underlyingpalisade cells, large and closely packed. Circular striations can beobserved. Those cells treated with the invented solution are lessobscured versus those treated with acidified chloral hydrate solution:FIG. 5A, fragment of upper epidermis underlying parenchyma cells,cuticle is irregular striated, using acidified chloral hydrate solution;FIG. 5B, fragment of upper epidermis underlying parenchyma cells,cuticle is irregular striated, using clearing agent and mountingsolution of Ex 1.

FIG. 6 shows a microscopic view of fragments or groups of pericycle offibers. Fibers are lignified, moderately thickened and have pitted walls(gap in the internal secondary thickening of the cell wall). Pits fromthe fibers in FIGS. 6A and 6B can be clearly observed with both clearingagent and mounting solutions: FIG. 6A, longitudinal view of a veinsection showing fibers which have thickened walls with rounded or slitshaped pit (gap in the internal secondary thickening of the cell wall),using acidified chloral hydrate solution; FIG. 6B, longitudinal view ofa vein section showing fibers which have thickened walls with rounded orslit shaped pit (gap in the internal secondary thickening of the cellwall), using clearing agent and mounting solution of Ex 1.

FIG. 7 shows a microscopic view of the lower epidermis showingcharacteristic anomocytic stomata. There was no difference between theclearing agent and mounting solutions in clarity and function: FIG. 7A,lower epidermis surface showing anomocytic stomata and circularcuticular striations, using acidified chloral hydrate solution; FIG. 7B,lower epidermis surface showing anomocytic stomata and circularcuticular striations, using clearing agent and mounting solution of Ex1.

Example 4 Safflower (Flower), Carthamus tinctorius L. (Asteraceae);FIGS. 8-9

Powdered samples of flowers from Safflower have the abundant pollengrains with three noticeable germinal pores. The exine (outer coat ofthe pollen grain) is dentate and spinose. The presence of laticferousducts with a reddish brown secretion next to vessels was observed. Inground samples of safflower, pollen grains with three germinal pores,exine and laticiferous ducts with a reddish-brown secretion next tovessels could be observed.

FIG. 8 shows a microscopic view of rounded or elliptical pollen grainswith three germinal pores, exine (outermost cell wall of pollen grain)dentate spinose: FIG. 8A, characteristic pollen grain with threegerminal pores, exine dentate, using acidified chloral hydrate solution;FIG. 8B, characteristic pollen grain with three germinal pores, exinedentate, using clearing agent and mounting solution of Ex 1.

FIG. 9 shows a microscopic view of laticiferous ducts (tubular cellscontaining latex fluid) with a reddish-brown secretion next to vesselselements. There were no differences observed using clearing agent andmounting solution, versus acidified chloral hydrate solution: FIG. 9A,two laticiferous ducts with a darker secretion next to the vesselelements, using acidified chloral hydrate solution; FIG. 9B,laticiferous duct filled with a darker secretion next to the vesselelements, using clearing agent and mounting solution of Ex 1.

FIG. 11 displays fresh whole mounted plant specimens cleared withExample 1 clearing agent and mounting solution. A-B: Basil leaf. A,epidermis with diacytic stomata, capitate and peltate glands; B:mesophyll cells with chloroplasts; C-F: Oregano leaf. C, coveringtrichomes with thick cell walls over the vein and capitate glands; D,Close up of capitate glands (arrow); E: depicting epidermis and peltateoil gland; F: mesophyll cells; G-H: Arabidopsis thaliana root; G, roottip cellular differentiation; H: xylem differentiation in root.

The clearing and mounting solution and its derivatives and/or analogscan also be used effectively in the same or a similar manner with cellsor tissues from animals including, without limitation, poultry, humans,livestock, reptiles, amphibians, insects and mites, as well as protists,mold, fungi, bacteria, and other microorganisms.

The specimens displayed in FIGS. 12-21 were prepared analogously tothose above, using the clearing and mounting solution of Example 1.

FIG. 12 shows a red alga Polysiphonia sp gametophyte showing a secondarybranch forming off the main axis.

FIG. 13 shows round worm free living nematode Panagrellus redivivus(Animalia) anterior end showing internal structures (buccal cavity andjuveniles hatched internally).

FIG. 14 shows a small aquatic crustacean Daphnia sp. (Animalia) showinganterior section

FIG. 15 shows characteristic Drosophila melanogaster (Animalia) compoundeye, showing numerous ommatidia (light detectors).

FIG. 16 shows a dorsal view of the head of Drosophila melanogaster(Animalia) showing compound eye, antenna and mouth parts.

FIG. 17 shows fungus Penicillium sp. conidiophores with a chain ofconidia (asexual spores) at the end.

FIG. 18 shows basil downy mildew (Peronospora belbahrii), protist, withdistinct staining of characteristic branched conidiophores and conidiaafter one week of inoculation. Stained with iodine solution and sulfuricacid.

FIG. 19 shows a round worm Panagrellus redivivus (Animalia) stained withfuchsine.

FIG. 20 shows characteristic starch grain of Ginger stained with iodinesolution.

FIG. 21 shows characteristic lignified fiber of Prunus africana stainedwith phloroglucinol/HCl solution.

The present invention has been described and exemplified with thespecific embodiments disclosed above, and in the following claims, whichare not intended to limit the scope of the invention in any way.

All references cited herein are incorporated by reference in theirentireties.

What is claimed is:
 1. A clearing agent and mounting solutioncomprising: (a) 10% to 97% (v/v) of trichloroethanol; (b) 0% to 5% (v/v)of trichloroacetic acid; (c) 12.5% to 50% (v/v) of glycerol; and (d)optionally, water; wherein the refractive index of said solution isgreater than or equal to 1.3810.
 2. The clearing agent and mountingsolution of claim 1, further comprising a C1-C6 alcohol.
 3. The clearingagent and mounting solution of claim 2, wherein said C1-C6 alcohol isselected from the group consisting of methanol, ethanol, and a mixturethereof.
 4. The clearing agent and mourning solution of claim 1, furthercomprising an acid selected from the group consisting of hydrochloricacid, sulfuric acid, phosphoric acid, and mixtures of two or morethereof.
 5. The clearing agent and mounting solution of claim 1, furthercomprising a dissolved plastic polymer or resin.
 6. The clearing agentand mounting solution of claim 1, further comprising monomeric units,the polymerization of which stabilizes the mounting solution, a specimenthat is mounted in the mounting medium or both.
 7. The clearing agentand mounting solution of claim 1, further comprising one or more C1-C12organic acids.
 8. The clearing agent and mounting solution of claim 1,further comprising a chemical preservative to preserve the integrity ofa specimen mounted in the mounting solution or to extend its working andviewable lifetime.
 9. The clearing agent and mounting solution of claim1, further comprising an alkaline hydroxide base.
 10. The clearing agentand mounting solution of claim 1, further comprising a compound selectedfrom the group consisting of ethylene glycol, polyethylene glycols andderivatives thereof.
 11. The clearing agent and mounting solution ofclaim 5, further comprising polyvinylpyrrolidone dissolved in a solventselected from the group consisting of ethanol, methanol, isopropanol,acetone and mixtures of two or more thereof.
 12. The clearing agent andmounting solution claim 1, further comprising dimethylsulfoxide.
 13. Theclearing agent and mounting solution of claim 1, further comprising anaromatic solvent selected from the group consisting of xylene, toluene,other benzene derivatives and mixtures of two or more thereof.
 14. Theclearing agent and mounting solution of claim 1, further comprising anaqueous solution of a carbohydrate selected from the group consisting ofmono-, di- and poly-saccharide forms of C4-C6 carbohydrates, andmixtures of two or more thereof.
 15. The clearing agent and mountingsolution of claim 1, wherein the trichloroacetic acid is present at 0.3%to 5% (v/v).
 16. The clearing agent and mounting solution of claim 1,wherein the trichloroethanol is present at 34.5% to 97%, thetrichloroacetic acid is present at 0.3% to 5%, and the glycerol ispresent at 0% to 12.5% (v/v).
 17. The clearing agent and mountingsolution of claim 2, wherein the said C1-C6 alcohol is present 5% to85%.
 18. The clearing agent and mounting solution of claim 3, whereinthe alcohol is methanol.
 19. The clearing agent and mounting solution ofclaim 3, wherein methanol and/or ethanol is/are present at 25% to 30%.20. The clearing agent and mounting solution of claim 17, wherein thetrichloroethanol is present at about 56.25% (v/v), the trichloroaceticacid is present at about 1.25% (v/v), the glycerol is present at about12.5% (v/v), and the C1-C6 alcohol is methanol, wherein the methanol ispresent at about 30% (v/v).
 21. A method of preparing a specimencomprising: a) soaking a specimen in a sufficient quantity of theclearing agent and mounting solution of claim 1 to provide a clearedspecimen, (b) applying a fluorescent stain or dye to the clearedspecimen to provide a cleared stained specimen, whereby the features ofthe specimen are selectively highlighted, (c) applying the clearedstained specimen to a microscope slide, cuvette, or well forobservation, (d) optionally, applying a cover slip over the specimen,and (e) optionally, visualizing the specimen using a microscope selectedfrom the group consisting of a fluorescent microscope, an epifluorescentmicroscope, a confocal microscope and combinations of two or morethereof.
 22. A method of preparing a specimen for microscopy comprising:(a) applying a specimen to be examined to a microscope slide; (b)applying a sufficient quantity of the clearing agent and mountingsolution of claim 1 to mount said specimen that is effective to increasethe transparency of said specimen and to allow multiple vertical planesto be visualized without the need to section, remount, or further modifysaid specimen; and (c) optionally, applying a cover slip over thespecimen.
 23. A method of preparing a specimen for microscopycomprising: (a) applying a specimen to be examined to a microscopeslide; (b) applying a sufficient quantity of the clearing agent andmounting solution of claim 1 to mount said specimen that is effective tosimultaneously dehydrate, de-pigment, and clear said specimen formicroscopic or visual analysis; and (c) optionally, applying a coverslip over the specimen.